REDUNDANCY AND FUNCTION OF RIBOSOMAL RNA GENES IN BACILLUS SUBTILIS

枯草芽孢杆菌核糖体RNA基因的冗余和功能

• 批准号: 6240177
• 负责人: RIVKA RUDNER
• 金额: $ 2.6万
• 依托单位: HUNTER COLLEGE
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-01-01 至 1997-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6240177
• 关键词: Bacillus subtilis; DNA directed RNA polymerase; Escherichia coli; beta galactosidase; fusion gene; gene expression; gene interaction; gene mutation; gene redundancy; genetic mapping; genetic promoter element; genetic transcription; microorganism genetics; microorganism growth; nucleic acid sequence; operon; plasmids; polymerase chain reaction; ribosomal RNA; spores; transfer RNA; western blottings

The proposal deals with the heterogeneity in organization of the conserved and redundant rRNA (rm) operons, their expression and the molecular mechanisms involved in the regulation of ribosome synthesis in the endospore-forming bacteria Bacillus subtilis and has three major goals: 1) To study the differential expression and growth rate control of the 10 rrn by creating single-copy lacZ fusion capable of integrating at the native rrn site or at heterologous loci (amyE, thrC). 2) To establish the role and interaction of the tandemly arranged promoters (P1,P2) and Upstream Activating Sequences (UAS) in growth rate regulation by studying the expression of promoter parts and after saturation mutagenesis in vital regions (-10, -35, -50, and -88) of various rrn operons as a function of different growth rates or during amino acid starvation in relA+ and relA- 3) To establish the role of specific rrn, RNA polymerase and the stringent control system (relA) in the life cycle of B. subtilis by inducing deletions or transposon (Tn917) insertions into selected rrn and isolating mutants defective in growth regulation and to ascertain how these mutants function during vegetative growth, sporulation and germination. The experimental approach involves recombinant DNA techniques of plasmid constructions, gene fusions to the E. coli lacZ gene, in vivo and in vitro mutagenesis, primer extension analysis, B-galactosidase measurements and assays of (p)ppGpp accumulation.

该提案涉及组织中的异质性 保守和冗余的rRNA（RM）操纵子，其表达和 参与调节核糖体合成的分子机制 枯草芽孢杆菌形成内孢子的细菌，有三个主要 目标：1）研究差异表达和增长率控制 通过创建能够整合的单拷贝Lacz融合，在10个RRN中 在本地RRN站点或异源基因座（Amye，THRC）。 2）到 建立串联布置的启动子的作用和相互作用 （P1，P2）和上游激活序列（UAS）在生长速率调节中 通过研究启动子部分的表达和饱和后 各种RRN的重要区域（-10，-35，-50和-88）的诱变 操纵子是不同生长速率或氨基酸期间的函数 Rela+和Rela- 3）在建立特定RRN的作用， 生命周期中的RNA聚合酶和严格的控制系统（RELA） 通过诱导缺失或转座子（TN917）插入的枯草芽孢杆菌 进入选定的RRN并隔离生长调节中有缺陷的突变体 并确定这些突变体在营养生长过程中的作用， 孢子和发芽。 实验方法涉及 质粒构建的重组DNA技术，基因融合到与 大肠杆菌lacz基因，体内和体外诱变，底漆延伸 分析，B-半乳糖苷酶测量和（P）PPGPP的测定 积累。